Immunoassay is a technique using an antibody for the analysis of a sample. In the early days of immunoassay, researchers mainly performed this technique to measure or detect a protein. But, recently with the advance of antibody-technique, immunoasaay has been widely performed to analyze low-molecular compounds, carbohydrates, lipids and many microorganisms. Among various immunoassays, the most preferred one is the sandwich enzyme-linked immunosorbent assay, which uses two different antibodies having different epitopes against one antigen. This method demonstrates high antigen selectivity, so that it has been largely used for diagnostic purpose.
To increase sensitivity in detection during immunoassay, methods for amplifying signals biochemically have been developed, for example IPCR (immuno-PCR) to amplify signals biochemically by using DNA and polymerase, Bio-barcode immunoassay to amplify signals chemically by using nanoparticles and DNA, cascade or proenzyme activation system to amplify signal biochemically (see FIG. 1, U.S. Pat. No. 4,463,090), etc. Regarding IPCR, it was reported that IPCR exhibited 100-10,000 times as high sensitivity as the typical immunoassay. However, this method has problems of poor reproducibility and quantitativeness. Besides, when polyclonal antibody is used, even the selectivity against antigen is decreased, suggesting that this method is not preferred for the application in biosamples (Brakmann S et al., Angew Chem Int Ed 43:5730-5734, 2004). Moreover, researchers continuously ask higher detection sensitivity (Yolken R H et al., J. Clin. Microbiol. 10:317-321, 1979).
Therefore, to increase detection sensitivity in enzyme-linked immunosorbent assay, the present inventors developed a novel cascade enzyme-linked immunoassay by linking a cascade reaction initiator to a nano-particle on which an antigen-specific antibody is immobilized.